The present invention concerns agents for prevention or decrease of damages caused by stress conditions, particularly ischemica and/or hypoxia and to pharmaceutical compositions comprising such agents.
Hypoxia, defined as lack or decrease of oxygen levels and ischemia, defined as lack or decrease of a blood supply, are common medical disorders of the vascular network, which may be caused by various reasons including: lack of oxygen such as in cases of drowning or suffocation, narrowing of blood vessels due to depositions on their walls, blockage of blood vessels for example by blood clots, and damage to blood vessels which can interpret their integrity. Decline or cessation of the normal blood supply to a specific tissue causes damage, which if prolonged can be irreversible leading to massive cell death. Where ischemia is caused due to the narrowing of coronary blood vessels and decrease of the blood supply to the heart, it may lead ultimately to myocardial infarction, one of the most common causes of mortality in the western world (Sabia, et al., N. Engl. J. Med., 327:1825-1831, (1992); Sasayama, S., Fujita, M., Circulation, 85:1197-1204, (1992)).
The myocardial vascular network is regulated to a great extent by microenvironmental induced hypoxia caused by decrease of blood flow, i.e. ischemia. The molecular mechanism of hypoxia-regulated gene expression of cell surface receptors, involved in the regulation and maintenance of the cardiovascular system, is poorly understood. Insufficient blood supply following restenosis is the leading cause of heart failure as a result of ischemic stress.
Hypoxia has been suggested as one of the microenvironmental factors that induce angiogenesis (Schaper, et al., Cir. Res., 28:671-679 (1971)) and modulate the phenotype of smooth muscle cells (Rugh, K. et al., Cardiovac. Res., 21:730-736 (1987); Shweiki, et al., Nature, 359:843-845 (1992)) and myocytes (Stenmark, et al., Chest., 93:127-133 (1988)). Hypoxic and ischemic stress causes a series of well documented changes in myocardial cells and tissues, including increased anaerobic glycolysis, loss of contractility and eventually cell death. In the heart, the induction of the proto-oncogenes fos and jun in cardiac myocytes exposed to severe hypoxia has been described (Konziolka, et al., J. Neuroser., 69:494-499 (1988)). This induction occurred after exposure of between 1 and 4 hours to hypoxia, and then declined, coinciding with loss of myocytes contractility but prior to irreversible cell damage.
Current attempts to treat or prevent myocardial damage due to hypoxia or ischemia involve the development of collateral circulation, an alternative source of blood supply to the myocardium, to provide adequate flow to the major epicardial branches of the coronary artery, which blood flow is prevented by failure of the original vessels. This may be achieved by the secretion of the angiogenic factor bFGF or the upregulation of VEGF under hypoxia. Thus, while cardiac myocytes undergo ischemia, collaterals may develop actively by growth, DNA replication and mitosis of endothelial and smooth muscle cell (SMC). Collateral development by heparin binding growth factors has the disadvantage that it may take a rather long period before collateral develops and functionality take over pre-existing blood vessels.
Thrombin is an agent having multiple effects on the cells of the vascular and circulation systems. In hemostasis thrombin has a central role as a serine protease that converts fibrinogen to fibrin which clots blood. Additional functions of thrombin are widespread and diverse and appear to involve cellular activations which are mediated through cellular thrombin receptor(s). For example, thrombin is the most potent activator of platelets; it is chemotactic for monocytes; it is mitogenic for lymphocytes and mesenchymal cells including vascular smooth muscle cells and; it promotes numerous responses within the vascular endothelium. (Coughlin, et al., J. Clin. Invest. 89:351-355 (1992)). Because these cell activating functions of thrombin occur within the range of concentrations normally required for the clotting of blood, thrombin has been proposed to play important physiological roles not only in hemostasis and thrmobosis but may also have principle roles in mediating responses to vascular injury such as leukocyte chemotaxis (to mediate inflammation), cellular proliferation (to mediate restenosis), glomerulonephritis, wound repair (such as occurs in bone remodelling), smooth muscle cell proliferation and ligation of xcex1vxcex23 integrin through a cryptic yet functional RGD site (Bar-Shavit, et al., J. Clin. Invest., 84:1096-1104 (1989); Bar-Shavit et al., Cell Reg., 1:453-463, (1990)).
The cellular receptor of thrombin (ThR) is preferentially upregulated in advanced atherosclerotic lesions following percutaneous transluminal coronary angioplasty. This receptor is present also in rate ventricular myocytes implicated in the physiological maintenance of these cells.
Thrombin receptor is a seven transmembrane G-coupled protein that belongs to a new family of receptors termed xe2x80x9cProtease Activated Receptorsxe2x80x9d (PAR). Unlike most growth factor receptors, the activation of PAR does not require the traditional ligand receptor complex formation. Instead, receptors of this family serve as substrates for protease digestion to yield an irreversible form of activated receptor that conveys further cellular effects. Activation of the ThR was found to be important in various physiological systems such as myocytes (Glenbotski et al., J. Biol. Chem., 268:20646-20652 (1993)), keratinocytes (Santulli et al., PNAS, 92:1-6 (1995)) and astrocyte proliferation (Grabham, P. and Cunningham, D., J. Neurochem., 64(2):583-591 (1995)). Other PARs have been discovered to have importance in diverse physiological systems (Coughlin et al., PNAS, 91; 9200-12, (1994)) an example being PAR-2 receptor which is involved in the inflammation cascade systems (Nystedt et al., PNAS, 91:9208-9212 (1994); Nystedt et al., Eur. J. Biochem., 232:84-89 (1995); Nystedt et al., J. Biol. Chem., 271:19910-19915 (1996); Schmidt, J. Biol. Chem., 271:9307-9212 (1996)).
The present invention is based on the surprising finding that mRNA levels of the thrombin receptor, a member of the Protease-Activated Receptor (PAR) family, decrease under conditions of stress, such as hypoxia. It has further been found that activating the ThR prior to or during the exposure to hypoxia, prevents said hypoxia-induced decreased in the level of mRNA. Administration of a ThR activating agents to cells under normal conditions, i.e. normal oxygen level, did not cause an increase in the normal levels of ThR mRNA levels.
ThR is a growth promoting receptor for cells of different origins, for example: fibroblasts, smooth muscle cells, astrocytes, etc. In myocytes activation of ThR induces hypertrophy and increases atrial natriuretic factor gene expression.
Therefore, maintaining a normal level of ThR mRNA is necessary for the well being and maintenance of cells in general and of myocytes in particular. Thus a decrease in the level of ThR mRNA under stress conditions, such as hypoxia contributes to the deterioration of cells, and particularly myocytes under these conditions. Agents capable of preventing such a stress-induced decrease in the level of ThR mRNA may help prevent or decrease some of the damages caused to the cells under stress conditions.
Thus, the present invention provides an agent for preventing the decrease in the levels of protease-activated receptor (PAR) mRNA under stress conditions, said agent being an activator of the PAR.
The term xe2x80x9cpreventing the decrease of mRNA levelsxe2x80x9d refers to a complete prevention of the decrease of these levels and reversal to normal mRNA levels featured by the same type of cells under normal conditions. Alternatively, this term refers to situations where there is partial prevention, i.e. while the mRNA level of the PAR in the cells which are under the stress condition treated by the activator of PAR, may not reach the level featured in those cells under normal (i.e. non-stress) conditions, it nevertheless is higher than corresponding mRNA levels of PAR in cells exposed to the same stress conditions and which are not treated by the activator of PAR.
The term xe2x80x9cProtease-Activated Receptorxe2x80x9d (PAR) refers to a family of G-protein coupled seven transmembranal receptors which are activated by proteolytic cleavage of their extracellular amino terminus. Examples of such PAR are the thrombin receptor, (Vu et al., Cell, 64:1057-1068 (1991)); and PAR-2; (Santulli et al., Cell Biol., 92:1-5, 1995)).
The term xe2x80x9cactivatorsxe2x80x9d refers to agents which are capable of causing an interaction of the PAR to the G-protein, for example, as determined by initiation of cell signaling. Cell signaling can be determined, for example, in the same manner as shown for the activation of the src family kinased, wherein Pertussis Toxin, a G-protein inhibitor, partially inhibits src-like kinase. Other modes of determining activation of G-protein seven transmembrane receptors are well known in the art. The mechanism of activation may be by cleavage of the extracellular amino terminus of the PAR receptor.
Examples of suitable activators which work by cleavage of the PAR are various proteases particularly serine proteases such as non-physiological trypsin, catepsin G, plasmin, etc. An example of a protease specifically suitable for ThR is either the native thrombin or xcex1-thrombin.
Alternatively, the activation can be achieved by providing analogues of the receptor cleavage products, i.e. the internal ligand (also termed xe2x80x9ctethered receptorxe2x80x9d) which are able to mimic a cleaved receptor and thus activate it. For example, thrombin receptor activating peptides (TRAP) are capable of mimicking the activated receptor by activating directly the second transmembrane-loop of the receptor, thereby by creating tethered ligand and cellular response.
Other activators of various PARs which work by mimicking the tethered receptor are well known in the art.
For ThR various peptides termed xe2x80x9cThrombin Receptor Activating Peptidesxe2x80x9d (TRAP) are used. Examples of TRAP are peptides selected from the following group:
-SFLLRNPNDKYEPF (SEQ ID NO: 1),
-SFLLRNPNDKYEP (SEQ ID NO: 2);
-SFLLRNPNDKYE (SEQ ID NO: 3);
-SFLLRNPNDKY (SEQ ID NO: 4);
-SFLLRNPNDK (SEQ ID NO: 5);
-SFLLRNPND (SEQ ID NO: 6);
-SFLLRNPN (SEQ ID NO: 7); and
-SFLLRNP (SEQ ID NO: 8).
Another type of activator acting as an analog of the cleavage product of a ThR isolated from Xenopus laevis is the peptide TFRIFP (SEQ ID NO: 13).
The murine protease activated receptor PAR-2 is activated by the peptide SLIGRL.
The term xe2x80x9cstress conditionsxe2x80x9d refers to conditions which are substantially different from normal or optimal conditions necessary for maintenance or growth of cells, tissue or organs without being so extreme so as to be immediately lethal, although exposure to such conditions for a prolonged period of time may eventually lead to massive cell-death. Examples of such stress conditions are: hypoxia, ischemia and hypoglycemia. Preferably, the stress conditions are hypoxia and/or ischemia which are connected. As explained above, ischemia actually leads eventually to hypoxia, since the tissue or organ is deprived of oxygen carrying blood.
The agent of the present invention may serve as an active ingredient in a pharmaceutical composition which purpose is to prevent or diminish damages to the cells, tissues or organs caused by stress conditions as specified above, in particular hypoxia and/or ischemia.
By a preferred embodiment the pharmaceutical composition is intended to prevent or diminish damage caused by stress conditions, due to decrease in the level of ThR mRNA, since normal levels of ThR mRNA are necessary for the maintenance of many cell types, notably myocytes.
Therefore the present invention further concerns a pharmaceutical composition for the treatment of cardial disorders caused by lack of oxygen or blood flow comprising a pharmaceutically acceptable carrier and as an active ingredient a therapeutically effective amount of an active agent according to the description as specified above.
Examples of such disorders are delayed ischemic heart failure, myocardial infarction, complete occlusion and angina pectoris.
The term xe2x80x9ctreatmentxe2x80x9d in the context of the present invention does not necessarily mean complete recovery but can also refer to a certain amount of prevention, or alleviation of some of the damages caused by the stress conditions, which preferably are hypoxia and/or ischemia.
Preferably, the cardial disorders are myocardial disorders.
Since not all the damage caused by hypoxia/ischemia is due to decrease of the level of ThR mRNA, but also to other mechanisms, the pharmaceutical composition of the invention should preferably be administered together with other agents or medicaments capable of minimizing damage caused to the cardial system by stress conditions.
The activation of a thrombin receptor has a variety of affects on the blood, vascular system, smooth muscle cells, etc., not all of which are beneficial. Therefore, it is preferable as much as possible to administer the ThR activator to the desired site directly and not systematically. Thus, in accordance with the preferred embodiment of the present invention the pharmaceutical compositions are administered directly to the heart, for example, by local reperfusion to the heart or by administering locally to the heart compounds capable of low and controlled release of the pharmaceutical composition.
Since the purpose of the pharmaceutical composition of the invention is to prevent or decrease cardial, especially myocardial damage caused by hypoxia and/or ischemia, it should be administered as close as possible to the initiation of the hypoxia and/or ischemic conditions.
The present invention will now be further illustrated with reference to some non-limiting examples.